1. Field of the Invention:
This invention relates to the measurement and quantification of lymphocyte subclasses or subpopulations. More particularly, the invention relates to a method and an apparatus for measuring, based on the principle of flow cytometry, blood cells to which fluorescently tagged monoclonal antibodies are bound for identifying lymphocyte subclasses.
2. Description of the Prior Art:
The white blood cells in the blood of a healthy individual include a variety of lymphocytes, monocytes and granulocytes (neutrophils, eosinophils and basophils). Among these, lymphocytes are composed of a plurality of subclasses such as B cells, T cells, N cells and K cells. The T cells can be further divided into such subtypes as helpers, suppressors and killer T cells. Measuring the relative numbers of these lymphocyte subclasses and constituent cells is useful in diagnosing immunological incompetence and in ascertaining the condition of various immunoregulatory disorders. It is also useful in observing the progress of medical treatment of various illnesses.
Each subclass of lymphocyte is characterized by an antigenic determinant on the surface of the cells belonging to the subclass. These antigenic determinants can be identified by specific monoclonal antibodies uniquely bound to the respective antigenic determinants. Examples of monoclonal antibodies for identifying T cells include CD2 (OKT11, Leu-5), CD3 (OKT3, Leu-4) and CD5 (OKT1, Leu-1). An example of a monoclonal antibody for identifying B cells is CD20 (B1). Further, an example of a monoclonal antibody for identifying T cells is CD4 (OKT4, Leu-3).
If these monoclonal antibodies are fluorescently tagged and reacted with blood cells and the fluorescence due to the fluorescent stain is examined, the lymphocytes that belong to a specific subclass can be detected.
A method of identifying and quantifying lymphocyte subclasses based on the principle of flow cytometry using fluorescently tagged antibodies is disclosed in e.g. the specification of Japanese Patent Application Laid-Open Publication (KOKAI) No. 56-16872. In order to identify lymphocytes in accordance with the disclosed method, signal processing is performed in which a two-dimensional signal strength distribution is displayed using signals on two channels, and a two-dimensional specific region in which lymphocytes are present is determined and enclosed.
A problem with the conventional method described above is that the signal processing for automatically determining and enclosing a specific region in the two-dimensional signal strength distribution in which the lymphocytes are present is extremely complicated and is not suited to an apparatus for identifying and quantifying lymphocyte subclasses automatically.
Though the construction of the apparatus and the procedure are greatly simplified if only one type of signal, e.g. side-scattered light only, is used to distinguish lymphocytes based on the signal strength distribution, isolating the lymphocytes from the ghosts of white or red blood cells is unsatisfactory.
The specification of Japanese Patent Application Laid-Open Publication (KOKAI) No. 60-36960 discloses a method of distinguishing lymphocytes among white and red blood cells by combining measured values of forward-scattered light and side-scattered light detected when blood cells are irradiated with light, and converting these measured values into a one-dimensional parameter. However, effecting the conversion into a one-dimensional parameter is a complicated task requiring a voluminous amount of hardware and software. It is also necessary to make the conversion one blood cell at a time, thus requiring an exorbitant amount of time to measure a single blood sample. As a result, the method is not practical for application to an automated apparatus.